Anthrax and Small Pox replikins and methods of use

ABSTRACT

Isolated peptides of the  Bacillus anthracis  Anthrax Toxin Lethal factor Protein pX01-107, antibodies specific for the peptides and methods of stimulating the immune response of a subject to produce antibodies to the  Bacillus anthracis  Anthrax Toxin Lethal factor Protein pX01-107 are disclosed. Also disclosed are isolated peptides of the Small Pox Virus Surface Antigen S Precursor Protein, antibodies specific for the peptides and methods of stimulating the immune response of a subject to produce antibodies to the Small Pox Virus Surface Antigen S Precursor Protein.

CROSS REFERENCE TO OTHER APPLICATIONS

[0001] This application claims priority to Provisional ApplicationsSerial Nos. 60/303,396 filed Jul. 09, 2001 and 60/278,761 filed Mar. 27,2001, and Continuation-in-Part applications Ser. Nos. 09/146,755 filedSep. 04, 1998 (issued as U.S. Pat. No. 6,242,578 B1) and 09/817,144filed Mar. 27, 2001 (pending), the latter of which claim priority toapplication Ser. No. 08/198,139 filed Feb. 17, 1994 (abandoned), whichare incorporated herein in their entirety by reference thereto.

FIELD OF THE INVENTION

[0002] This invention relates to the identification and use ofReplikins, a class of peptides that share structural characteristics. Inparticular, this invention relates to Replikins which have beenidentified in Bacillus anthracis and Small Pox Virus (Variola).

BACKGROUND OF THE INVENTION

[0003] Glycoprotein 10B is a membrane glycoprotein isolated from brainglioblastoma multiforme, lymphoma and breast cancer cells (U.S. Pat. No.6,242,578 B1). A constituent peptide of Aglyco 10B, malignin, isenriched in cell membranes tenfold during anaerobic replication.Hydrolysis and mass spectrometry of malignin yielded a 16-mer peptideincluding (SEQ ID NO.: 1) kagvaflhkk. This peptide, which is absent fromthe normal human genome, was assumed to be acquired.

SUMMARY OF THE INVENTION

[0004] In one aspect of the invention there are provided isolatedBacillus anthracis (Anthrax) peptides containing a replikin sequence.The Anthrax peptides comprise from 7 to about 50 amino acids including(1) at least one lysine residue located six to ten amino acid residuesfrom a second lysine residue; (2) at least one histidine residue; and(3) at least 6% lysine residues. In another embodiment of this aspect ofthe invention there are provided Small Pox Virus peptides containing areplikin sequence which comprises from 7 to about 50 amino acidsincluding (1) at least one lysine residue located six to ten amino acidresidues from a second lysine residue; (2) at least one histidineresidue; and (3) at least 6% lysine residues.

[0005] In another aspect of the invention there is provided a processfor stimulating the immune system of a subject to produce antibodiesthat bind specifically to Anthrax polypeptides containing a replikinsequence, said process comprising administering to the subject aneffective amount of a dosage of a composition comprising at least oneAnthrax replikin peptide. In a preferred embodiment the compositioncomprises at least one peptide selected from SEQ ID NO. 91, SEQ ID NO.92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ IDNO. 79, SEQ ID NO. 98 or a combination thereof.

[0006] In another embodiment of this aspect of the invention there isprovided a process for stimulating the immune system of a subject toproduce antibodies that bind specifically to Small Pox Viruspolypeptides containing a replikin sequence, said process comprisingadministering to the subject an effective amount of a dosage of acomposition comprising at least one Small Pox Virus replikin peptide. Ina preferred embodiment the composition comprises a peptide selected fromSEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ IDNO. 103, or a combination thereof.

[0007] In another aspect of the invention there are provided antisensenucleic acid molecules complementary to the coding strand of the gene orto the mRNA encoding the Bacillus anthracis Anthrax Lethal FactorProtein pX01-107 peptide, wherein said antisense nucleic acid moleculeis complementary to a nucleotide sequence encoding the peptide of SEQ IDNO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98.

[0008] There are also provided antisense nucleic acid moleculecomplementary to the coding strand of the gene or to the mRNA encodingthe Small Pox Virus Surface Antigen S Precursor Protein, wherein saidantisense nucleic acid molecule is complementary to a nucleotidesequence encoding the peptide of SEQ ID NO. 99, SEQ ID NO. 100, SEQ IDNO. 101, SEQ ID NO. 102, or SEQ ID NO. 103.

[0009] As used herein, the term “peptide” refers to a compound of two ormore amino acids in which the carboxyl group of one is united with anamino group of another, forming a peptide bond. The term peptide is alsoused to denote the amino acid sequence encoding such a compound. Thus, apeptide sequence may be a subsequence of a larger polypeptide sequence.As used herein, a Replikin peptide is peptide consisting essentially of7 to about 50 amino acid including (1) at least one lysine residuelocated six to ten amino acid residues from a second lysine residue; (2)at least one histidine residue; and (3) at least 6% lysine residues.Similarly, a replikin sequence is the amino acid sequence encoding sucha peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 is a bar graph depicting the frequency of occurrence ofreplikins in various protein groups.

[0011]FIG. 2 is a graph depicting the percentage of malignin permilligram total membrane protein during anaerobic replication ofglioblastoma cells.

[0012]FIG. 3 is a bar graph showing amount of antimalignin antibodyproduced in response to exposure to the recognin 16-mer.

[0013]FIG. 4A is a photograph of a blood smear taken with ordinary andfluorescent light. FIG. 4B is a photograph of a blood smear taken withand fluorescent light illustrating the presence of two leukemic cells.FIG. 4C is a photograph of a dense layer of glioma cells in the presenceof antimalignin antibody. FIG. 4D and FIG. 4E are photographs of thelayer of cells in FIG. 4C taken at 30 and 45 minutes following additionof antimalignin antibody.

[0014]FIG. 4F is a bar graph showing the inhibition of growth of smallcell lung carcinoma cells in vitro by antimalignin antibody.

[0015]FIG. 5 is a plot of the amount of antimalignin antibody present inthe serum of patients with benign or malignant breast disease pre-andpost surgery.

[0016]FIG. 6 is a box diagram depicting an embodiment of the inventionwherein a computer is used to carry out the 3-point-recognition methodof identifying replikin and recognin sequences.

DETAILED DESCRIPTION OF THE INVENTION

[0017] In one aspect of the invention there is provided a method foridentifying nucleotide or amino acid sequences that include a recogninor replikin sequence. The method is referred to herein as a3-point-recognition method. By use of the “3-point recognition” method,described herein below, a new class of peptides was revealed in algae,yeast, fungi, amoebae, bacteria, plant and virus proteins havingreplication, transformation, or redox functions. This class of peptidesis referred to herein as replikins.

[0018] One example of a replikin that was identified by the3-point-recognition method, is the amino acid sequence, (SEQ ID NO.: 2)hsikrclgiifdk, which occurs in Saccharomyces cerevisiae “replicationbinding protein”. Five replikins were found in amino acids 1-163 of the“replicating protein” of tomato leaf curl Gemini vinis. Amino acids1-160 of this tomato virus protein bind DNA. Another replikin, (SEQ IDNO.: 3) hkqkivapvk, is highly conserved in 236 isolates of foot andmouth disease virus.

[0019] Although replikins were found to be present in only 1.5% ofpublished sequences identified by the PubMed data bank as “viruspeptides” as a whole, and in only 8.5% of sequences identified as “brainpeptides” plus “neuropeptides”, surprisingly, replikins were found in100% of “tumor viruses”, in 85% of “transforming proteins,” and 97% of“cancer proteins” (as categorized in the PubMed data bank). Therecognin, (SEQ ID NO.: 4) ykagvaflhkkndide, was not found in publishedsequences of the human genome.

[0020] The 16-mer recognin peptide, (SEQ ID NO.: 4) ykagvaflhkkndide,when synthesized and injected as vaccine into a mammal, has been shownto produce antimalignin antibody, which is cytotoxic to malignantreplicating cells of several types at picogram per cell amounts.Replikins identified in organisms such as diatom plankton, H. pylori,tomato leaf curl virus, foot and mouth disease virus, hepatitis B and Cviruses, and HIV, also are thus targets for diagnosis and treatment oras vaccines for the control of replication of their respective virussource.

[0021] Table 1 illustrates the sequence of the malignin peptide, the16-mer recognin sequence, (SEQ ID NO.: 4) ykagvaflhkkndide. TABLE 116-mer peptide sequence ykagvaflhkkndide obtained from malignin byhydrolysis and mass spectrometry Method By Which Fragment Obtained Auto-Auto- hydrolysis of hydrolysis malignin Seq of malignin immobilized IDFragment MH+ free in on bromoace- Microwaved Microwaved NO. Identified(mass) Sequence solution tyl cellulose 5 seconds 30 seconds 19 1-3381.21 ( )yka(g) + 20 1-5 537.30 ( )ykagv(a) + 21 2-6 445.28(y)kagva(f) + 22 2-7 592.35 (Y)kagvaf(l) + 23  4-11 899.55(a)gvaflhkk(n) + 24 5-7 336.19 (g)vaf(l) + 25 6-7 237.12 (v)af(l) + 26 6-10 615.36 (v)aflhk(k) + 27  6-10 615.36 (v)aflhk(k) + 28  6-12 857.50(v)aflhkkn(d) + 29  6-12 857.50 (v)afhkkn(d) + 30 7-8 279.17 (a)fl(h) +31 10-16 861.43 (h)kkndide( ) + 32 11-14 489.27 (k)kndi(d) + 33 12-15476.2- (k)ndid(e) +

[0022] The malignin peptide was isolated from membranes of glioblastomamultiforme (glioma) cells grown in tissue culture (U.S. Pat. No.6,242,578 B1). The sequence of a 16-mer peptide of malignin wasdetermined by hydrolysis and mass spectrometry: (SEQ ID NO.: 4)ykagvaflhkkndide (Table 1). A search of published human genome sequencesfor sequence encoding the 16-mer amino acid sequence was negative. Sincethis 16-mer peptide was absent from normal human genome data a searchwas made of sequences of other organisms for possible origins andhomologues. No identical sequences were found. But, using the sequenceof the 16-mer peptide as a template, and constructing a“3-point-recognition” method to visually scan protein sequences ofseveral different organisms, a new class of peptides, the replikins, wasrevealed in organisms as diverse as algae, yeast and viruses.Surprisingly, these peptides were found to be concentrated in larger‘replicating ’ and ‘transforming’ proteins (so designated by theirinvestigators, based on activities, see Table 2).

[0023] Table 2 illustates several replikin sequences that wereidentified by the 3-point-recognition method of the invention. TABLE 2Examples of replikins in various organisms - prototype: Glioma replikin*kagvaflhkk (SEQ ID No.: 1) Algae: SEQ ID 34 Caldophera proliferakaskftkh NO. 35 Isolepisprolifera kaqaetgeikgh Yeast: 36Schizoscceharomyces pombe ksfkypkkhk 37 Oryza sativa kkaygnelhk 2 Sacch.cerevisiae replication binding protein hsikrelgiifdk Fungi: 38Isocitrate lyase ICI 1, Penicillium marneffei kvdivthqk 39 DNA-dependentRNA polymerase 11, Diseula dcstructiva kleedaayhrkk 40 Ophiostomanovo-u1m 1, RNA in Dutch elm disease fungus kvilplrgnikgiffkh Amoeba: 41Entamoeba invadens, histone H2B klilkgdlnkh Bacteria: 42 Pribosomalprotein replication factor, Helicobacter pylori ksvhaflkReplication-associated protein Staph aureus 10 Mycoplasma pulmonic,chromosome replication kkektthnk 43 Macrophage infectivity potentiator,L. legionella kvhffqlkk 90 Bacillus anthracis kihlisvkk 91 Bacillusanthracis hvkkekeknk 92 Bacillus anthracis khivkievk 93 Bacillusanthracis kkkkikdiygkdallh 94 Bacillus anthracis kwekikqh 95 Bacillusanthracis kldqipppiepkkddiih 96 Bacillus anthracishnryasnivesayllilnewkn- niqsdlikk 97 Bacillus anthracishavddyagylldknqsdlvt- nskk 98 Bacillus anthracis haerlkvqknapk Plants:44 Arabidopsis thaliana, prolifera kdhdfdgdk 45 Arabidopsis thaliana,cytoplasinic ribosomal kmkglkqkkah 46 Arabidopsis thaliana, DNA bindingprotein kelssttqeksh Viruses: 9 Replication associated protein A [Maizestreak virus] kekkpskdeimrdiish 11 Bovine herpes virus 4, DNAreplication protein hkinitngqk 12 Meleagrid herpesvirus 1, replicationbinding protein hkdlyrllmk 47 Feline immunodeficiency hlkdyklvk 3 Footand Mouth Disease (O) hkqkivapvk 5 HIV Type 1 kcfncgkegh 7 HIV Type 2kcwncgkegh 99 Small Pox Virus (Variola) khynnitwyk 100 Small Pox Virus(Variola) kysqtgkeliih 101 Small Pox Virus (Variola) hyddvrikndivvsrck102 Small Pox Virus (Variola) hrfklildski 103 Small Pox Virus (Variola)kerghnyyfek Tumor 48 Rous sarcoma virus tryrosine-protein kinase kklrhekViruses: 49 v-yes, avian sarcoma kklrhdk 50 c-yes, colon cancer,malignant melanoma kklrhdk 51 v-srcC., avian sarcoma kklrhek 52 c-src,colon, mammary, panrcreatic cancer kklrhek 53 Neuroblastoma RAS viral(v-ras) oncogene kqahelak 54 VPl (major capsid protein) [Polyamavirussp.] kthrfskh 55 Sindbis knlhekik 56 El [Human papilloamavirus type 71]khrpllqlk 57 v-erbB from AEV and c-erb kspnhvk 58 v-fms (feline sarcoma)knihlekk 59 c-fms (acute and chronic myelomonocytic tumors) knihlekk 60large t-antigen I [Polyomavinis sp.1 kphlaqslek 61 middle t-antigen[Polyomavirus sp,1- kqhrelkdk 62 small t-antigen [Polyomavirus spJ,kqhrelkdk 63 v-abl, murine acute leukemia kvpvlisptlkh 64 Human T-celllymphotropic virus typo 2 kslllevdkdish 65 c-kit, GI tumors, small celllung carcinoma kagitimvkreyh 18 Hepatitis C hyppkpgcivpak Trans- 66Transforming protein myb ksgkhlgk forming 67 Transforming protein myc,Burkitt lymphoma krreqlkhk Proteins: 68 Ras-related GTP-binding proteinksfevikvih 69 Transforming protein ras (teratocarcinoma) kkkhtvkk 70TRAF-associated NF•kB activator TANK kaqkdhlsk 71 RFP transformingprotein hlkrvkdlkk 72 Transforming protein D (S.C.) kygspkhrlik 73Papilloma virus type 11, transforming protein klkhilgkarfik 74 Proteintryosine kinase (EC 2.7.1.112slk kgdhvkhykirk 75 Transforming protein(axl(−)) keklrdvmvdrhk 76 Transforming protein (N-myc) klqarqqqllkkieh77 Fibroblast growth factor 4 (Kaposi sarcoma) kkgnrvsptmkvth Cancer 78Matrix metaloproteinase 7 (uterine) keiplhfrk Cell 79 Transcriptionfactor 7-like kkkphikk Proteins: 80 Breast cancer antigen NY-BR-87ktrhdplak 81 BRCA-1-Associated Ring Domain Protein (breast) khhpkdnlik82 ‘Autoantigen from a breast tumor’ khkrkkfrqk 83 Glioma replikin (thisstudy) kagvaflhkk 84 Ovarian cancer antigen khkrkkfrqk 85 EE L leukemiakkkskkhkdk 86 Proto-oncogene tyrosine-protein kinase C-ABLE hksekpalprk87 Adenomatosis polyposis coli kkkkpsrlkgdnek 88 Gastric cancertransforming protein ktkkgnrvsptmkvth 89 Transforming protein (K-RAS2B), lung khkekmskdgkkkkkksk

[0024] Identifacation of an amino acid sequence as a replikin or ascontaining a replikin, i.e., a homologue of the malignin 16-mer peptide,requires that the three following “3-point recognition” requirements bemet. The peptide sequence must have (1) at least one lysine residuelocated six to ten residues from another lysine residue; (2) at leastone histidine residue; and (3) a composition of at least 6% lysinewithin an amino acid sequence of 7 to about 50 residues.

[0025] Databases were searched using the National Library of Medicinekeyword “Pub Med” descriptor for protein sequences containing replikinsequences. Sequences of all individual proteins within each group ofPubMed-classified proteins were visually scanned for peptides meetingthe three above-listed requirements. An infrequent occurrence ofhomologues was observed in “virus peptides” as a whole (1.5%), and inother peptides not designated as associated with malignanttransformation or replication such as “brain peptides” and“neuropeptides” (together 8.5%). Surprisingly, homologues wereidentified in 100% of “tumor viruses”, in 85% of “transformingproteins”, and in 97% of “cancer cell proteins” (FIG. 1). The peptidesidentified by this search were named replikins, and a ten amino acidportion of the 16-mer peptide, (SEQ ID NO.: 1) “kagvaflhkk”, was namedthe glioma replikin.

[0026] To permit classification of subtypes of replikins, additional or“auxiliary specifications” to the basic “3-point-recognition”requirements may be added: (a) on a structural basis, such as the commonoccurrence of adjacent di- and polylysines in cancer cell proteins(e.g., Transforming protein P2B(K-RAS 2B), lung, Table 2, SEQ ID NO.:89), and other adjacent di-amino acids in TOLL-like receptors, or b) ona functional basis, such as exhibiting ATPase, tyrosine kinase or redoxactivity as seen in Table 2.

[0027] Whether replikin structures are conserved or are subject toextensive natural mutation was examined by scanning the proteinsequences of various isolates of foot and mouth disease virus (FMDV),where mutations in proteins of these viruses have been well documentedworldwide for decades. Protein sequences of FMDV isolates were visuallyexamined for the presence of both the entire replikin and each of thecomponent replikin amino acid residues observed in a particularreplikin. For example, in the protein VP1 of FMDV type O, the replikin(SEQ ID NO.: 3) “hkqkivapvk” was found to be conserved in 78% of the 236isolates reported in PubMed, and each amino acid was found to beconserved in individual isolates as follows: his, 95.6% ; lys, 91.8%;gln 92.3%; lys, 84.1%; ile, 90.7%; val, 91.8%; ala, 97.3%; pro, 96.2%;ala, 75.4%; and lys, 88.4%. The high rate of conservation suggestsstructural and functional stability of the replikin structure.Similarly, sequence conservation was observed in different isolates ofHIV for its replikins, such as (SEQ ID NO.: 5) “kcfncgkegh” or (SEQ IDNO.: 6) “kvylawvpahk” in HIV Type 1 and (SEQ ID NO.: 7) “kcwncgkegh” inHIV Type 2 (Table 2). Other examples of conservation are seen in theconstant presence of malignin in successive generations, over 10 yearsof tissue culture of glioma cells, and by the constancy of affinity ofthe glioma replikin for antimalignin antibody isolated byimmunoadsorption from 8,090 human sera from the U.S., U.K., Europe andAsia (e.g., FIG. 5 and U.S. Pat. No. 6,242,578 B 1).

[0028] As seen in FIG. 2, during anaerobic respiration when the rate ofcell replication is increased, malignin is enriched. That is, maligninis found to increase not simply in proportion to the increase in cellnumber and total membrane proteins, but is enriched as much as tenfoldin concentration, starting with 3% at rest and reaching 30% of totalmembrane protein. This clear demonstration of a marked increase inreplikin concentration with glioma cell replication points to and isconsistent with the presence of replikins here sought by the 3-pointrecognition method and found in the proteins of various organisms whichwere found by mutation studies and other previous studies to be criticalto replication. For example, replikins were identified in such proteinsas “Saccharomyces cerevisiae replication binding protein” (SEQ ID NO.:2) (hsikrelgiifdk); the “replication associated protein A of maizestreak virus” (SEQ ID NO.: 8) (kyivcareahk and (SEQ ID NO.: 9)kekkpskdeimrdiish); the “replication-associated protein ofStaphylococcus aureus” (SEQ ID NO.: 10) (kkektthnk); the “DNAreplication protein of bovine herpes virus 4” (SEQ ID NO.: 11)(hkinitngqk); and the “Mealigrid herpes virus 1 replication bindingprotein” (SEQ ID NO.: 12) (hkdlyrllmk). Previous studies of tomato leafcurl gemini virus show that the regulation of virus accumulation appearsto involve binding of amino acids 1-160 of the “replicating protein” ofthat virus to leaf DNA and to other replication protein molecules duringvirus replication. Analysis of this sequence showed that amino acids1-163 of this “replicating protein” contain five replikins, namely: (SEQID NO.: 13) kfrinaknyfltyph, (SEQ ID NO.: 14) knletpvnklfiricrefh, (SEQID NO.: 15) hpniqaaksstdvk, (SEQ ID NO.: 16) ksstdvkaymdkdgdvldh, and(SEQ ID NO.: 17) kasalnilrekapkdfvlqfh.

[0029] Table 2 shows that replikin-containing proteins also areassociated frequently with redox functions, and protein synthesis orelongation, as well as with cell replication. The association withmetal-based redox functions, the enrichment of the replikin-containingglioma malignin concentration during anaerobic replication, and thecytotoxicity of antimalignin at low concentrations (picograms/cell)(FIG. 4c-f), all suggest that the replikins are related to centralrespiratory functions, which are perhaps less often subjected to themutations characteristic of proteins of more superficial location orless central survival function.

[0030] Of particular interest were eight different replikins identifiedin Bacillus anthracis, the organism responsible for anthrax infections;and five different replikins identified in small pox virus. The eightBacillus anthracis peptides are present in the Anthrax Toxin LethalFactor Protein pX01-107 and have the amino acid sequence of SEQ ID NO.91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ IDNO. 96, SEQ ID NO. 97 and SEQ ID NO. 98, respectively. The five smallpox virus peptides are present in the Small Pox Virus Surface Antigen SPrecursor Protein, which purportedly enhances Small Pox Virusreplication. The five peptides have the amino acid sequence of SEQ IDNO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102 and SEQ ID NO.103, respectively.

[0031] Data on anti-replikin antibodies support replikin class unity. Ananti-replikin antibody response has been quantified by immunoadsorptionof serum antimalignin antibody to immobilized malignin (see Methods inU.S. Pat. No. 5,866,690). The abundant production of antimaligninantibody by administration to rabbits of the synthetic version of the16-mer peptide whose sequence was derived from malignin, absentcarbohydrate or other groups, has established rigorously that thispeptide alone is an epitope, that is, it is a sufficient basis for thisimmune response (FIG. 3). The 16-mer peptide produced both IgM and IgGforms of the antibody. Antimalignin antibody was found to be increasedin concentration in serum in 37% of 79 cases in the U.S. and Asia ofhepatitis B and C, early, in the first five years of infection, longbefore the usual observance of liver cancer, which develops aboutfifteen to twenty-five years after infection. Relevant to bothinfectious hepatitis and HIV infections, transformed cells may be oneform of safe haven for the virus: prolonging cell life and avoidingvirus eviction, so that the virus remains inaccessible to anti-viraltreatment.

[0032] A synthetic replikin vaccine such as the glioma replikin (SEQ IDNO.: 1) “kagvaflhkk” or the hepatitis C replikin (SEQ ID NO.: 18)“hyppkpgcivpak”, or HIV replikins such as (SEQ ID NO.: 5) “kcfncgkegh”or (SEQ ID NO.: 6) “kvylawvpahk” may be used to augment antibodyconcentration in order to lyse the respective virus infected cells andrelease virus extracellularly where chemical treatment can then beeffective. Recognin and/or replikin peptides may be administered to asubject to induce the immune system of the subject to produceanti-replikin and/or anti-recognin antibodies. Generally, a 0.5 to about2 mg dosage, preferably a 1 mg dosage of each peptide is administered tothe subject to induce an immune response. Subsequent dosages may beadministered if desired.

[0033] In another embodiment of the invention, isolated recognin orreplikin peptides may be used to generate antibodies. Various proceduresknown in the art may be used for the production of antibodies toreplikin sequences or recognin sequences. Such antibodies include butare not limited polyclonal, monoclonal, chimeric, humanized, singlechain, Fab fragments and fragments produced by an Fab expressionlibrary. Antibodies that are linked to a cytotoxic agent may also begenerated.

[0034] For the production of antibodies various host animals may beimmunized by injection with a replikin or recognin peptide, includingbut not limited to rabbits, mice, rats, and larger mammals. Variousadjuvants may be used to enhance the immunological response, dependingon the host species, including but not limited to Freund's (complete andincomplete), mineral gels, such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, key limpet hemocyanin, dintrophenol, and potentiallyuseful human adjuvants such as BCG and Corynebacterium parvum.

[0035] Monoclonal antibodies to replikins or recognins may be preparedby using any technique that provides for the production of antibodymolecules by continuous cell lines in culture. These include but are notlimited to the hybridoma technique originally described by Kohler andMilstein, (Nature, 1975, 256:495-497), the human B-cell hybridomatechnique (Kosbor et al., 1983, Immunology Today, 4:72), and the EBVhybridoma technique (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniquesdeveloped for the production of chimeric antibodies (Morrison et al.,1984, Proc. Nat. Acad. Sci USA, 81:6851-6855) may be used.Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce replikin-or recognin-specific single chain antibodies.

[0036] Particularly useful antibodies of the invention are those thatspecifically bind to replikin sequences contained in peptides and/orpolypeptides of Bacillus anthracis. For example, antibodies to any ofpeptides SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98 and combinationsof such antibodies are useful in the treatment and/or prevention ofanthrax.

[0037] Similarly, antibodies to peptides SEQ ID NO. 99, SEQ ID NO. 100,SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103 and combinations of suchantibodies are useful in the treatment and/or prevention of small pox.

[0038] Antibody fragments which contain binding sites for a replikin orrecognin may be generated by known techniques. For example, suchfragments include but are not limited to F(ab′)₂ fragments which can beproduced by pepsin digestion of the antibody molecules and the Fabfragments that can be generated by reducing the disulfide bridges of theF(ab′)₂ fragments. Alternatively, Fab expression libraries can begenerated (Huse et al., 1989, Science, 246:1275-1281) to allow rapid andeasy identification of monoclonal Fab fragments with the desiredspecificity.

[0039] The fact that antimalignin antibody is increased in concentrationin human malignancy regardless of cancer cell type (FIG. 5), and thatthis antibody binds to malignant cells regardless of cell type now maybe explained by the presence of the replikin structures present in mostmalignancies (FIG. 1 and Table 2). Population studies have shown thatantimalignin antibody increases in concentration in healthy adults withage, and more so in high-risk families, as the frequency of cancerincreases. An additional two-fold or greater antibody increase whichoccurs in early malignancy has been independently confirmed with asensitivity of 97% in breast cancers 1-10 mm in size. Shown to localizepreferentially in malignant cells in vivo, histochemically the antibodydoes not bind to normal cells but selectively binds to (FIG. 4a,b) andis highly cytotoxic to transformed cells in vitro (FIG. 4c-f). Since inthese examples the same antibody is bound by several cell types, thatis, brain glioma, hematopoietic cells (leukemia), and small cellcarcinoma of lung, malignant replikin class unity is again supported.

[0040] Antimalignin does not increase with benign proliferation, butspecifically increases only with malignant transformation andreplication in breast in vivo and returns from elevated to normal valuesupon elimination of malignant cells (FIG. 5). Antimalignin antibodyconcentration has been shown to relate quantitatively to the survival ofcancer patients, that is, the more antibody, the longer the survival.Taken together, these results suggest that antireplikin antibodies maybe a part of a mechanism of control of cell transformation andreplication. Augmentation of this immune response may be useful in thecontrol of replication, either actively with synthetic replikins asvaccines, or passively by the administration of anti-replikinantibodies, or by the introduction of non-immune based organic agents,such as for example, carbohydrates, lipids and the like, which aresimilarly designed to target the replikin specifically. For organismssuch as diatom plankton, foot and mouth disease virus, tomato leaf curlgemini virus, hepatitis B and C, and HIV, and malignant cells,identified constituent replikins are useful as vaccines, and also may beusefully targeted for diagnostic purposes.

[0041] The replikin sequence structure is associated with the functionof replication. Thus, whether the replikins of this invention are usedfor targeting sequences that contain replikins for the purpose ofdiagnostic identification, promoting replication, or inhibiting orattacking replication, for example, the structure-function relationshipof the replikin is fundamental. Thus, while the structure of thereplikin may be a part of a larger protein sequence, which may have beenpreviously identified, it is necessary to utilize only the specificreplikin structure when seeking to induce antibodies that will recognizeand attach to the replikin fragment and thereby cause destruction of thecell. Even though the larger protein sequence may be known in the art ashaving a “replication associated function,” vaccines using the largerprotein often have failed or proven ineffective, even though theycontain one or more replikin sequences.

[0042] Although the present inventors do not wish to be held to a singletheory, the studies herein suggest that the prior art vaccines areineffective because they are based on the use of the larger proteinsequence. The larger protein sequence invariably has one or moreepitopes (independent antigenic sequences that can induce specificantibody formation); replikin structures usually comprise one of thesepotential epitopes. The presence of other epitopes within the largerprotein may interfere with adequate formation of antibodies to thereplikin, See, e.g., Webster, R. G., J. Immunol., 97(2):177-183 (

[0043] and Webster et al., J. Infect. Dis., 134:48-58, 1976; Klenermanet al, Nature 394:421-422 (1998) for a discussion of the well-knownphenomenon “original antigenic sin”). The formation of an antibody to anon-replikin epitope may allow binding to the cell, but not necessarilylead to cell destruction.

[0044] It is well known in the art that in the course of antibodyproduction against a “foreign” protein, the protein is first hydrolyzedinto smaller fragments. Usually fragments containing from about six toten amino acids are selected for antibody formation. Thus, if hydrolysisof a protein does not result in replikin-containing fragments,anti-replikin antibodies will not be produced. In this regard, it isinteresting that replikins contain lysine residues located six to tenamino acids apart, since lysine residues are known to bind to membranes.

[0045] Furthermore, replikin sequences contain at least one histidineresidue. Histidine is frequently involved in binding to redox centers.Thus, an antibody that specifically recognizes a replikin sequence has abetter chance of inactivating or destroying the cell in which thereplikin is located, as seen with anti-malignin antibody, which isperhaps the most cytotoxic antibody yet described, being active atpicograms per cell.

[0046] One of the reasons that vaccines directed towards a particularprotein antigen of a disease causing agent have not been fully effectivein providing protection against the disease (such as foot and mouthvaccine which has been developed against the VP1 protein or largesegments of the VP1 protein) is that antibody to the replikins have notbeen produced. That is, either epitopes other than replikins present inthe larger protein fragments may interfere according to the phenomenonof “original antigenic sin”, and/or because the hydrolysis of largerprotein sequences into smaller sequences for processing to produceantibodies results in loss of integrity of any replikin structure thatis present, e.g., the replikin is cut in two and/or the histidineresidue is lost in the hydrolytic processing. The present studiessuggest that for an effective vaccine to be produced, the replikinsequences, and no other epitope, should be used as the vaccine. Forexample, a vaccine of the invention can be generated using any one ofthe replikin peptides identified by the three point recognition system.Particularly preferred peptides for an anthrax vaccine include peptidesSEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO.95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, and combinationsthereof . Preferred peptides for use as a small pox vaccine are peptidesSEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ IDNO. 103 and combinations thereof. These peptides, alone or in variouscombinations are administered to a subject, preferably by i.v. orintramuscular injection, in order to stimulate the immune system of thesubject to produce antibodies to the peptide. Generally the dosage ofpeptides is in the range of from about 0.1 μg to about 10 mg, preferablyabout 10 μg to about 1 mg, and most preferably about 50 μg to about 500μg. The skilled practitioner can readily determine the dosage and numberof dosages needed to produce an effective immune response.

[0047] Replikin or recognin DNA or RNA may have a number of uses for thediagnosis of diseases resulting from infection with a virus, bacteriumor other replikin or recognin encoding agent. For example, replikin orrecognin nucleotide sequences may be used in hybridization assays ofbiopsied tissue to diagnose the presence of a particular organism, e.g.,Southern or Northern analysis, including in situ hybridization assays.

[0048] Also within the scope of the invention are oligoribonucleotidesequences, that include antisense RNA and DNA molecules and ribozymesthat function to inhibit the translation of replikin- orrecognin-containing mRNA. Both antisense RNA and DNA molecules andribozymes may be prepared by any method known in the art. The antisensemolecules can be incorporated into a wide variety of vectors fordelivery to a subject. The skilled practitioner can readily determinethe best route of delivery, although generally i.v. or i.m. delivery isroutine. The dosage amount is also readily ascertainable.

[0049] Particularly preferred antisense nucleic acid molecules are thosethat are complementary to a mRNA encoding a Bacillus anthracispolypeptide comprising a replikin sequence comprising from 7 to about 50amino acids including (1) at least one lysine residue located six to tenresidues from a second lysine residue; (2) at least one histidineresidue; and (3) at least 6% lysine residues. More preferred areantisense nucleic acid molecules that are complementary to the codingstrand of the gene or to the mRNA encoding the Bacillus anthracisAnthrax Lethal Factor Protein pX01-107 peptide, wherein the antisensenucleic acid molecule is complementary to a nucleotide sequence encodingthe peptide of SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ I) NO.94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. SEQ ID NO. 97, or SEQ IDNO. 98.

[0050] Another preferred set of antisense nucleic acid moleculesincludes those that are complementary to a mRNA encoding a Small PoxVirus polypeptide comprising a replikin sequence comprising from 7 toabout 50 amino acids including (1) at least one lysine residue locatedsix to ten residues from a second lysine residue; (2) at least onehistidine residue; and (3) at least 6% lysine residues. More preferredare antisense nucleic acid molecules that are complementary to thecoding strand of the gene or to the mRNA encoding the Small Pox VirusSurface Antigen S Precursor Protein, wherein the antisense nucleic acidmolecule is complementary to a nucleotide sequence encoding the peptideof SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, or SEQID NO. 103.

[0051] In another embodiment of the invention, immune serum containingantibodies to one or more replikin obtained from an individual exposedto one or more replikins may be used to induce passive immunity inanother individual or animal. Immune serum may be administered via i.v.to a subject in need of treatment. Passive immunity also can be achievedby injecting a recipient with preformed antibodies to one or morereplikins. Passive immunization may be used to provide immediateprotection to individuals who have been exposed to an infectiousorganism. Administration of immune serum or preformed antibodies isroutine and the skilled practitioner can readily ascertain the amount ofserum or antibodies needed to achieve the desired effect.

[0052] Visual scanning of over three thousand sequences was performed indeveloping the present 3-point-recognition methods. However, data bankscomprising nucleotide and/or amino acid sequences can also be scanned bycomputer for the presence of sequences meeting the 3 point recognitionrequirements.

[0053] The three point recognition method may also be modified toidentify other useful compounds of covalently linked organic molecules,including other covalently linked amino acids, nucleotides,carbohydrates, lipids or combinations thereof. In this embodiment of theinvention a sequence is screened for subsequences containing three ormore desired structural characteristics. In the case of screeningcompounds composed of covalently linked amino acids, lipids orcarbohydrates the subsequence of 7 to about 50 covalently linked unitsshould contain (1) at least one first amino acid, carbohydrate or lipidresidue located six to ten residues from a second of the first aminoacid, carbohydrate or lipid residue; (2) at least one second amino acid,lipid or carbohydrate residue; and (3) at least 6% of the first aminoacid, carbohydrate or lipid residue. In the case of screening nucleotidesequences, the subsequence of about 21 to about 150 nucleotides shouldcontain (1) at least one first amino acid residue located withineighteen to thirty nucleotides from a second codon encoding the firstamino acid residue; (2) at least one second amino acid residue; and (3)encodes at least 6% of said first amino acid residue.

[0054] According to another embodiment of the invention, the methodsdescribed herein may be performed by a computer. FIG. 6 is a blockdiagram of a computer available for use with the foregoing embodimentsof the present invention. The computer may include a processor, aninput/output device and a memory storing executable program instructionsrepresenting the 3-point-recognition methods of the foregoingembodiments. The memory may include a static memory, volatile memoryand/or a nonvolatile memory. The static memory conventionally may be aread only memory (“ROM”) provided on a magnetic, or an electrical oroptical storage medium. The volatile memory conventionally may be arandom access memory (“RAM”) and may be integrated as a cache within theprocessor or provided externally from the processor as a separateintegrated circuit. The non-volatile memory may be an electrical,magnetic or optical storage medium.

EXAMPLE 1

[0055] Process For Extraction, Isolation and Identifacation of Replikinsand the use of Replikins to Targets Label or Destroy Replikin-containingOrganisms

[0056] a) Algae

[0057] The following algae were collected from Bermuda water sites andeither extracted on the same day or frozen at −20 degrees C andextracted the next day. The algae were homogenized in a cold room (at 0to 5 degrees C) in 1 gram aliquots in neutral buffer, for example 100cc. of 0.005M phosphate buffer solution, pH7 (“phosphate buffer”) for 15minutes in a Waring blender, centrifuged at 3000 rpm, and thesupernatant concentrated by perevaporation and dialyzed againstphosphate buffer in the cold to produce a volume of approximately 15 ml.The volume of this extract solution was noted and an aliquot taken forprotein analysis, and the remainder was fractionated to obtain theprotein fraction having a pK range between 1 and 4. The preferred methodof fractionation is chromatography as follows:

[0058] The extract solution is fractionated in the cold room (4 degreesC) on a DEAE cellulose (Cellex-D) column 2.5×1.0 cm, which has beenequilibrated with 0.005M phosphate buffer. Stepwise eluting solventchanges are made with the following solutions:

[0059] Solution 1—4.04 g. NaH2PO4 and 0.5 g NaH2PO4 are dissolved in 15liters of distilled water (0.005 molar, pH7);

[0060] Solution 2—8.57 g. NaH2PO4 is dissolved in 2,480 ml. of distilledwater;

[0061] Solution 3—17.1 g. of NaH2PO4 is dissolved in 2480 ml ofdistilled water (0.05 molar, pH 4.7);

[0062] Solution 4—59.65 g. of NaH2PO4 is dissolved in 2470 ml distilledwater (0.175 molar);

[0063] Solution 5—101.6 g. of NaH2PO4 is dissolved in 2455 ml distilledwater (pH 4.3);

[0064] Solution 6—340.2 g. of NaH2PO4 is dissolved in 2465 of distilledwater (1.0 molar, pX-i 4.1);

[0065] Solution 7—283.63 g. of 80% phosphoric acid (H3P04) is made up in2460 ml of distilled water (1.0 molar, pH 1.0).

[0066] The extract solution, in 6 to 10 ml volume, is passed onto thecolumn and overlayed with Solution 1, and a reservoir of 300 ml ofSolution 1 is attached and allowed to drip by gravity onto the column.Three ml aliquots of eluant are collected and analyzed for proteincontent at OD 280 until all of the protein to be removed with Solution 1has been removed from the column. Solution 2 is then applied to thecolumn, followed in succession by Solutions 3, 4, 5, 6 aid 7 until allof the protein which can, be removed with each Solution is removed fromthe column. The eluates from Solution 7 are combined, dialyzed againstphosphate buffer, the protein content determined of both dialysand anddialyzate, and both analyzed by gel electrophoresis. One or two bands ofpeptide or protein of molecular weight between 3,000 and 25,000 Daltonsare obtained in Solution 7. For example the algae Caulerpa mexicana,Laurencia obtura, Cladophexa prolifera, Sargassum natans, Caulerpaverticillata, Halimeda tuna, and Penicillos capitatus, after extractionand treatment as above, all demonstrated in Solution 7 eluates sharppeptide bands in this molecular weight region with no contaminants.These Solution 7 proteins or their eluted bands are hydrolyzed, and theamino acid composition determined. The peptides so obtained, which havea lysine composition of 6% or greater are Replikin precursors. TheseReplikin peptide precursors are then determined for amino acid sequenceby hydrolysis and mass spectrometry as detailed in U.S. Pat. No.6,242,578 B1. Those which fulfill the criteria defined by the“3-point-recognition” method are identified as Replikins. This procedurecan also be applied to obtain yeast, bacterial and any plant Replikins.

[0067] b) Virus

[0068] Using the same extraction and column chromatography separationmethods as above in a) for algae, Replikens in virus-infected cells areisolated and identified.

[0069] c) Tumor cells in vivo and in vitro Tissue Culture

[0070] Using the same extraction and column chromatography separationmethods as above in a) for algae, Replikins in tumor cells are isolatedand identified. For example, Replikin precursors of Astrocytin isolatedfrom malignant brain tumors, Malignin (Aglyco 10B) isolated fromglioblastoma tumor cells in tissue culture, MCF7 mammary carcinoma cellsin tissue culture, and P₃J Lymphoma cells in tissue culture each treatedas above in a) yielded Replikin precursors with lysine content of 9.1%,6.7%, 6.7%, and 6.5% respectively. Hydrolysis and mass spectrometry ofAglyco 10B as described in Example 10 U.S. Pat. No. 6,242,578 B1produced the amino acid sequence, ykagvaflhkkndiide the 16-mer Replikin.

EXAMPLE 2

[0071] As an example of diagnostic use of Replikins: Aglyco 10B or the16-mer Repliken may be used as antigen to capture and quantify theamount of its corresponding antibody present in serum for diagnosticpurposes are as shown in FIGS. 2,3,4 and 7 of U.S. Pat. No. 6,242,578B1,

[0072] As an example of the production of agents to attach to Replikinsfor labeling, nutritional or destructive purposes: Injection of the16-mer Replikin into rabbits to produce the specific antibody to the16-mer Replikin is shown in Example 6 and FIGS. 9A and 9B of U.S. Pat.No. 6,242,578 B1.

[0073] As an example of the use of agents to label Replikins: The use ofantibodies to the 16-mer Replikin to label specific cells which containthis Replikin is shown in FIG. 5 and Example 6 of U.S. pat. No.6,242,578 B1.

[0074] As an example of the use of agents to destroy Replikins: The useof antibodies to the 16-mer Replikin to inhibit or destroy specificcells which contain this Replikin is shown in FIG. 6 of U.S. Pat. No.6,242,578 B1.

[0075] From a proteomic point of view the construction of a “3-pointrecognition” template based on the new glioma peptide sequence leddirectly to identification of a biology-wide class of proteins havingrelated structures and functions. The operation of the3-point-recognition method resembles identification by the use of a“keyword”search; but instead of using the exact spelling of the keyword“kagvafihkk” as in a typical sequence homology search, or in thenucleotide specification of an amino acid, an abstraction of the keyworddelimited by the “3-point-recognition” parameters is used. Thisdelimited abstraction, although derived from a single relatively shortamino acid sequence leads to identification of a class of proteins withstructures that are defined by the same specifications. That particularfunctions, in this case transformation and replication, in addition tostructures, turn out also to be shared by members of the exposed classsuggests that these structures and functions are related. Thus, fromthis newly identified short peptide sequence, a molecular recognition‘language’ has been formulated, which previously has not been described.Further, the sharing of immunological specificity by diverse members ofthe class, as here demonstrated for the cancer replikins, suggests thatB cells and their product antibodies recognize replikins by means of asimilar recognition language. Since “3-point-recognition” is a proteomicmethod that specifies a particular class of proteins, using three ormore different recognition points for other peptides similarly shouldprovide useful information concerning other proteins classes. Further,the “3-point-recognition” method is applicable to other recognins, forexample to the TOLL ‘innate’ recognition of lipopolyssacharides oforganisms.

[0076] Several embodiments of the present invention are specificallyillustrated and described herein. However, it will be appreciated thatmodifications and variations of the present invention are encompassed bythe above teachings and within the purview of the appended claimswithout departing from the spirit and intended scope of the invention.

What is claimed is:
 1. An isolated Bacillus anthracis peptide comprisingfrom 7 to about 50 amino acids including (1) at least one lysine residuelocated six to ten residues from a second lysine residue; (2) at leastone histidine residue; and (3) at least 6% lysine residues.
 2. Thepeptide of claim 1 wherein the peptide has the amino acid sequence asset forth in SEQ ID NO.
 91. 3. The peptide of claim 1 wherein thepeptide has the amino acid sequence as set forth in SEQ ID NO.
 92. 4.The peptide of claim 1 wherein the peptide has the amino acid sequenceas set forth in SEQ ID NO.
 93. 5. The peptide of claim 1 wherein thepeptide has the amino acid sequence as set forth in SEQ ID NO.
 94. 6.The peptide of claim 1 wherein the peptide has the amino acid sequenceas set forth in SEQ ID NO.
 95. 7. The peptide of claim 1 wherein thepeptide has the amino acid sequence as set forth in SEQ ID NO.
 96. 8.The peptide of claim 1 wherein the peptide has the amino acid sequenceas set forth in SEQ ID NO.
 97. 9. The peptide of claim 1 wherein thepeptide has the amino acid sequence as set forth in SEQ ID NO.
 98. 10.An isolated small pox virus peptide comprising from 7 to about 50 aminoacids including (1) at least one lysine residue located six to tenresidues from a second lysine residue; (2) at least one histidineresidue; and (3) at least 6% lysine residues.
 11. The peptide of claim10 wherein the peptide has the amino acid sequence as set forth in SEQID NO.
 99. 12. The peptide of claim 10 wherein the peptide has the aminoacid sequence as set forth in SEQ ID NO.
 100. 13. The peptide of claim10 wherein the peptide has the amino acid sequence as set forth in SEQID NO.
 101. 14. The peptide of claim 10 wherein the peptide has theamino acid sequence as set forth in SEQ ID NO.
 102. 15. The peptide ofclaim 10 wherein the peptide has the amino acid sequence as set forth inSEQ ID NO.
 103. 16. An antibody that specifically binds to a Bacillusanthracis peptide or polypeptide sequence comprising from 7 to about 50amino acids including (1) at least one lysine residue located six to tenamino acid residues from a second lysine residue; (2) at least onehistidine residue; and (3) at least 6% lysine residues.
 17. An antibodycocktail comprising a plurality of antibodies, wherein each of saidantibodies specifically binds to a Bacillus anthracis peptide orpolypeptide sequence comprising from 7 to about 50 amino acids including(1) at least one lysine residue located six to ten amino acid residuesfrom a second lysine residue; (2) at least one histidine residue; and(3) at least 6% lysine residues.
 18. An antibody that specifically bindsto a small pox virus peptide or polypeptide sequence comprising from 7to about 50 amino acids including (1) at least one lysine residuelocated six to ten amino acid residues from a second lysine residue; (2)at least one histidine residue; and (3) at least 6% lysine residues. 19.An antibody cocktail comprising a plurality of antibodies, wherein eachof said antibodies specifically binds to a small pox virus peptide orpolypeptide sequence comprising from 7 to about 50 amino acids including(1) at least one lysine residue located six to ten amino acid residuesfrom a second lysine residue; (2) at least one histidine residue; and(3) at least 6% lysine residues.
 20. A method of stimulating the immunesystem of a subject to produce antibodies to Bacillus anthraciscomprising administering an effective amount of at least one Bacillusanthracis peptide comprising from 7 to about 50 amino acids including(1) at least one lysine residue located six to ten amino acid residuesfrom a second lysine residue; (2) at least one histidine residue; and(3) at least 6% lysine residues.
 21. The method of claim 18 comprisingadministering at least one peptide selected from the group consisting ofSEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO.95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO.
 98. and combinationsthereof.
 22. A method of stimulating the immune system of a subject toproduce antibodies to small pox virus comprising administering aneffective amount of at least one small pox virus peptide comprising from7 to about 50 amino acids including (1) at least one lysine residuelocated six to ten amino acid residues from a second lysine residue; (2)at least one histidine residue; and (3) at least 6% lysine residues. 23.The method of claim 20 wherein the at least one peptide is selected fromthe group consisting of SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101,SEQ ID NO. 102, SEQ ID NO. 103 and combinations thereof.
 24. Anantisense nucleic acid molecule complementary to a mRNA encoding aBacillus anthracis polypeptide comprising a replikin sequence comprisingfrom 7 to about 50 amino acids including (1) at least one lysine residuelocated six to ten residues from a second lysine residue; (2) at leastone histidine residue; and (3) at least 6% lysine residues.
 25. Anantisense nucleic acid molecule complementary to the coding strand ofthe gene or to the mRNA encoding the Bacillus anthracis Anthrax LethalFactor Protein pX01-107 peptide, wherein said antisense nucleic acidmolecule is complementary to a nucleotide sequence encoding the peptideof SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ IDNO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO.
 98. 26. An antisensenucleic acid molecule complementary to a mRNA encoding a Small Pox Viruspolypeptide comprising a replikin sequence comprising from 7 to about 50amino acids including (1) at least one lysine residue located six to tenresidues from a second lysine residue; (2) at least one histidineresidue; and (3) at least 6% lysine residues.
 27. An antisense nucleicacid molecule complementary to the coding strand of the gene or to themRNA encoding the Small Pox Virus Surface Antigen S Precursor Protein,wherein said antisense nucleic acid molecule is complementary to anucleotide sequence encoding the peptide of SEQ ID NO. 99, SEQ ID NO.100, SEQ ID NO. 101, SEQ ID NO. 102, or SEQ ID NO. 103.